The biology of reconstitution of T cell populations following acute loss remains incompletely characterized. Using murine models, we first identified two primary pathways of T cell immune reconstitution, the classic, thymic-dependent pathway, and a second, thymic-independent pathway. We then identified T cell surface markers which allowed identification, by phenotyping of reconstituted T cell populations, of the pathways which had given rise to them, and then applied this information to the characterization of T cell reconstitution in patients. Initial work established the applicability of this approach to the study of T cell population regeneration in humans who were young. Recent work verified validity of the approach and established the course of T cell immune reconstitution in adult humans over an extended period time for each of the two primary pathways. This work showed an essential role for the thymus in regenerating CD4+ T cells quantitatively. CD8+ T cells can numerically be reconstituted by peripheral expansion for immune reconstitution, but both CD4+ and CD8+ T cells require thymic activity for maintenance or regeneration of repertoire diversity. These findings have led in turn to a research emphasis on understanding mechanisms which control thymic function, and new treatments, including vaccine strategies, to treat cancer in the setting of a regenerating immune system. Four models of thymic regulation have been developed; this work has progressed to the developoment of two new project areas of research -- one focused on points of regulation of thymus function and one on introducing agents into clinical trials. The work addressed in this project has also led to efforts in investigating IL-7 effects on the maturation of thymocytes. We have identified IL-7 as a negative regulator of thymopoiesis as well as being essential for thymocyte development. These dual roles are dose dependent and negative regulation at higher concentrations is mediated through control of Notch signaling -- which is central to T/B lineage commitment. Additionally, we have worked to identify genes which might regulate the thymus, and have characterized a gene called Tbata (previously SPATIAL) which is a negative regulator acting within the stromal cell compartment. It appears to exert its effect through control of cell cycle, specifically through regulation of the Nedd8 pathway. Recent results indicate that regulation of cell cycle by Tbata extends beyond Nedd8 and involves p53. The cellular and molecular mechanisms by which androgen signaling blockade and IGF-1 modulate thymus function were characterized and map points of regulation of proliferation to the epithelial cell compartment of the thymus. A new transgenic murine model has been developed to investigate signaling pathways involved in this regulation. This new model involves the ability to deplete the peripheral T cell compartment without ancillary tissue injury. Using the information that a key control point of thymus regulation is the control of thymic epithelial cell proliferation, gene arrays are being carried out at fixed time intervals on isolated cells to identify activated cell pathways of division. In parallel, LC/MS is being used to identify new protein species that may act as ligands for those identified pathways. Three unidentified proteins have been found and are being validated. The role of IL-7 as a possible regulator of thymus function was noted above; its role in peripheral homeostasis has been further investigated by charactreizing IL-7 receptor regulation among T cell subsets. The purpose in all of these studies is to understand the biology of T cell homeostasis in order to develop new approaches to therapy for patients in whom T cell populations are depleted with consequences of impaired immunity. To date, IL-7 has been introduced in humans and a clinical trial with androgen blockade is in progress. The trial with androgen blockage shows early positive results which need to be confirmed as the trial continues. The work with IL-7 receptor modulation has shown a complex pattern of differential signaling regulation among subsets with the apparent effect of favoring the sustaining of primitive naive T cells. This is of special interest because it provides a basis for understanding how a single cytokine such as IL-7 can differentially regulate multiple distinct T cell subsets. Specifically, there is differential regulation between naive CD4 T cells versus recent thymic emigrants, and also between CD4 and CD8 T cells. Differences in signaling distal to the receptor for IL-7 appear to mechanistically account for this differential regulation. In humans, we noted that CD4 recovery was more limited than CD8 recovery following chemotherapy. Reasons for this are not known. We are now addressing the biology of signaling by the two primary homeostatic cytokines in the CD4 versus the CD8 subsets in murine models and have found a new layer of complexity in signaling that remains to be characterized. We have also made the observation that in T cell immune reconstitution reliant on the thymus-independent pathway in humans, that such expansion results in premature aging of the T cells with resultant cellular senescence, a likely contributor to T cell functional impairment following transplant.